Infectious Bursal Disease Virus antigenic isolates and vaccines

ABSTRACT

Antigenic isolates and vaccines for Infectious Bursal Disease Virus include variants of the molecular Group 6 family of IBDV isolates, in particular the 28-1 isolate.

This application is a continuation of U.S. application Ser. No.11/076,713, filed on Mar. 10, 2005, which claims the benefit under 35U.S.C. §119(e) to U.S. Provisional Application No. 60/552,989, filedMar. 12, 2004, which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel antigenic isolates of InfectiousBursal Disease Virus, as well as to vaccine compositions containing oneor more of these isolates. The invention also relates to new methods forpreventing or ameliorating Infectious Bursal Disease in poultry.

BACKGROUND OF THE INVENTION

Infectious Bursal Disease, also called “Gumboro Disease,” is an acuteand highly contagious viral infection of young chickens and other fowl.It is caused by Infectious Bursal Disease Virus (IBDV) Type I, which isa member of a group of viruses called Birnaviridae. The disease ischaracterized by degeneration of lymphoid tissue. The primary target ofinfection is the bursa of Fabricius, although lymphoid damage may alsooccur in the spleen, thymus, and gland of Harder.

Degeneration of the bursa of Fabricius and other lymphoid tissue inyoung chickens has severe economic consequences, as the infectedchickens have a decreased response to vaccination and an increasedsusceptibility to other infectious agents such as Newcastle disease,Marek's disease and infectious bronchitis disease. Poultry producers canlose a significant portion of their flock due to IBDV infection. Oftentimes, mortality rates can approach 80% or more in young chickens.

Immunization is the principal method for controlling the disease.Chickens may be passively immunized, by receiving maternally-derivedantibodies, or they may be actively immunized with live,live-attenuated, or killed (inactivated) vaccines. Live-attenuatedvaccines contain the virus that has been “modified” or attenuatedthrough serial passaging in cell culture. By passaging it is hoped toproduce a virus strain that is less pathogenic. In order to be useful ina vaccine, however, it must retain the antigenic and immunogenicproperties of the original virus. It must, that is, induce theproduction of neutralizing antibodies. Control of the disease byimmunization had been largely successful until variant strains began toemerge as the result of antigenic drift under field conditions. Thesevariants were causing disease in both actively and passively immunizedchickens.

Infectious Bursal Disease Virus is often separately classified in theart as either Standard or “STC” strain, or variant types, although thevast majority of wild-type IBDVs in the United States are now variants.Delaware viruses are some of the most common variant types, and DelawareE in particular has been considered the prototypical variant type foryears. IBDV surveys indicate that Delaware E virus is still commonlyisolated; however, other variant virus types such as GLS, Rs593 and AL2have also become quite prevalent. These strains can be characterizedusing a panel of monoclonal antibodies. In addition, using PCR-RFLPtechniques, a distinct molecular class of viruses (called Group 6) haverisen in prevalence over the past several years. Amino acid sequencingof this family of viruses shows that most are distinct from theprototype Delaware-E virus in the region generally regarded as perhapsone of the most critical to antigenic identity or uniqueness.

U.S. Pat. No. 5,919,461 relates to a live, variant vaccine strain whichis reportedly effective in immunizing young chickens against theStandard, the Delaware and other new-type variant strains.

U.S. Pat. No. 6,471,962 discloses the use of certain monoclonalantibodies in the diagnosis, prevention and treatment of InfectiousBursal Disease.

U.S. Pat. No. 5,192,539 relates to an IBDV vaccine for poultry withantigen material which is derived from a mammalian cell line.

U.S. Pat. No. 5,804,195 provides a vaccine for preventing infectiousbursal disease which contains specific strains of live, attenuated butintermediately virulent IBDV. The vaccine may contain other poultryimmunogens, including those against Newcastle Disease virus, Marek'sdisease virus, and infectious bronchitis virus.

In addition, WO 9105569 is related to a diagnostic and vaccine whichutilizes an IBDV variant with altered recognition sites.

There currently exists a need in the art for better antigenic isolatesof IBDV, as well as vaccines which comprise these isolates for useagainst IBDV infection. Also needed are better methods of protectingpoultry, in particular chickens, from the many variants of the IBDvirus, including newer ones that have recently emerged.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a vaccine composition whichis effective in preventing or ameliorating Infectious Bursal DiseaseVirus infection, which comprises an antigen or antigenic componenthaving the substantive identifying characteristics of an InfectiousBursal Disease Virus antigen as set forth in FIG. 1. As that term isused herein, “substantive identifying characteristics” means the aminoacid sequence of isolate 28-1 in FIG. 1, which comprises only onesubstitution G->D at position 318, i.e. 318-D, in major hydrophilic peakB of the Viral Protein-2 (VP-2) of IBDV, and in addition, one or more ofthe other substitutions noted for isolate 28-1 in FIG. 1, including thefollowing: 222-T, 249-K, 254-S or N, 279-N and 286-I. In addition, theantigen or antigenic component should retain substantial reactivity witha basic panel of monoclonal antibodies that characterizes Delawarestrain variant viruses.

Thus, the invention provides a vaccine composition comprising an antigenor antigenic component having a 318-D as the only substitution in majorhydrophilic peak B of the VP-2 amino acid sequence of Infectious BursalDisease Virus.

The invention is also directed to a vaccine composition againstInfectious Bursal Disease Virus, which comprises the novel 28-1antigenic isolate of IBDV deposited on Mar. 4, 2004 with the ATCC underAccession Number PTA-5848.

In a further embodiment of the invention there is provided an InfectiousBursal Disease Virus antigenic isolate having a 318-D as the onlysubstitution in major hydrophilic peak B of the VP-2 amino acid sequencewhich is suitable for use in a vaccine against IBD.

The invention also provides an Infectious Bursal Disease Virus antigenicisolate having the amino acid sequence set forth in FIG. 1 (28-1). Thisis a unique isolate in what is now referred to as the molecular Group 6family of IBDV isolates.

In addition, there is provided an Infectious Bursal Disease Virusantigenic isolate deposited on Mar. 4, 2004 with the ATCC underAccession Number PTA-5848 as part of the invention.

Also provided is a method for inducing protection against infection fromInfectious Bursal Disease Virus, which involves administering to apoultry animal a vaccine composition containing an antigen having thesubstantive identifying characteristics of an IBDV antigenic isolate asset forth in FIG. 1.

The invention further provides a method for inducing protection againstInfectious Bursal Disease, which comprises administering to a poultryanimal an antigenic isolate identified with ATCC Accession # PTA-5848.

These and other embodiments, features and advantages of the inventionwill become apparent from the detailed description and the appendedclaims set forth herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an amino acid sequence comparison of several reference U.S.variant IBDVs to classic (standard) virus. The “Consensus” sequenceshown in FIG. 1 is represented by SEQ ID NO:1, and the VP-2 variabledomains of STC, Del-E, Rs593, GLS and 28-1 are represented by SEQ IDNOs: 2 through 6, respectively.

FIG. 2 depicts the Jackwood PCR-RFLP testing system showing theprevalence of wild-type viruses in the United States over time. 1997data is taken from Jackwood survey. 1998-2003 data is taken from FortDodge surveys of over 600 positive samples.

FIG. 3 depicts the percent protection levels observed in a progenychallenge study, field trial #1, using a vaccine composition of theinvention and a commercial vaccine.

FIG. 4 depicts the percent protection levels observed in a progenychallenge study, field trial #2, using a vaccine composition of theinvention and a commercial vaccine.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention is directed to a novel InfectiousBursal Disease Virus (IBDV) antigenic isolate. The isolate is generallycharacterized as a Delaware-type virus using monoclonalantibody-antigen-capture ELISA, in that it reacts the same as Delaware-Eagainst a limited panel of monoclonal antibodies. In addition, the virusis further characterized using the reverse transcriptase-polymerasechain reaction/restriction fragment length polymorphism (RT-PCR/RFLP)method of Jackwood et al., Restriction Fragment Length Polymorphisms inthe VP2 Gene of Infectious Bursal Disease Viruses, Avian Dis.41:627-637, 1997, as a molecular Group 6 virus (rather than molecularGroup 2 to which the prototype Delaware-E variant is assigned).

Referring to FIG. 1, further characterization in terms of the amino acidsequence of the variable region of the major antigenic Viral Protein 2(VP-2) is set forth, along with sequence information from establishedisolates for comparison. Also included is a summary of thecharacterization of IBDV isolates with particular attention to thespecial qualities of the 28-1 isolate as a suitable example (hereinbelowdefined). Those skilled in the art will recognize that VP-2 is one ofthe two major structural proteins of IBDV and is also the target of theserotype-specific neutralizing antibodies which confer protectiveimmunity. (The second major structural protein of the virus, VP-3, doesnot elicit neutralizing antibodies.)

As FIG. 1 illustrates, IBDV isolates have two major and two minorhydrophilic peak regions. Without being bound by theory, all thehydrophilic regions, and in particular the major hydrophilic regions,are believed to have an important effect on antigenicity. The 28-1isolate has 318-D as its sole substitution in peak B noted in FIG. 1,and is thus separately identifiable from other reference IBDV variants,including the classic (standard—STC) strain and the Delaware-E strain.In fact, the 28-1 isolate differs from Delaware-E in what is generallyregarded as the most critical antigenic region of IBDV—major hydrophilicpeak B. In addition, the 28-1 isolate of IBDV has one or more of thefollowing additional substitutions: 222-T, 249-K, 254-S or N, 279-N and286-1 (as per commonly accepted single letter amino acid abbreviations).In addition, the 28-1 isolate differs from the Delaware-A variant atposition 222. Delaware-A has 222-Q which is a relatively raresubstitution in this position, whereas the 28-1 has 222-T in itspreferred embodiment. Most field viruses have 222-T, which perhapsindicates that the 28-1 isolate is a better antigenic match thanDelaware-A for U.S. field viruses, and also significant enough todistinguish the two strains as well. A summary of the amino acidpatterns in the virus strains presented in FIG. 1 is set forth in TABLE1 below:

TABLE 1 COMPARISON OF REFERENCE VIRUS AMINO ACID PATTERNS IN CRITICALVP-2 POSITIONS. Virus 222 249 254 286 318 321 322 323 Classic-STC P Q GT G A G D Delaware E T K S I D A G E GLS T K S T G E G D Rs593 T K N I GA E D 28-1 T K S I D A G D Delaware A Q K S I D A G D Positions 318,321, 322 and 323 are located in Peak B and amino acid changes result inantigenic change.

Each of the positions above in TABLE 1 has been linked to the binding ofneutralizing monoclonal antibodies, and thus the skilled artisan can seethe differences between the strains, including the Group 6 28-1 isolate.

Accumulation of data via PCR typing of U.S. field samples has nowclearly shown the prevalence and significance of the molecular Group 6IBDV viruses. Group 6 viruses are by far the most common molecular typerecovered from U.S. broilers, accounting for approximately 1 in 3 fieldisolates, as FIG. 2 indicates. Without being bound by any theory, theincreasing prevalence of Group 6 viruses in U.S. broilers since 1997 islikely a result of more successful escape from Delaware-E and Classictype antibodies provided by conventional IBD vaccines. In support ofthis concept is the fact that sequencing analysis of several U.S. fieldsamples shows that most IBD viruses in the Group 6 family do not havethe Delaware-E substitution pattern in the major hydrophilic PeakB—Jackwood et al., Amino Acid Comparison of Infectious Bursal DiseaseViruses Placed in the Same and Different Molecular Groups byRT/PCR-RFLP, Avian Dis. 45:330-339, 2001.

The practical significance of the Group 6 family of viruses was recentlyexplored by conducting a progeny challenge study of five breeder flockson five different killed IBD vaccination programs. One of Jackwood'sGroup 6 variants was compared against Delaware E and GLS strain virusesin a progeny challenge study. Results showed that the Group 6 variantvirus broke through maternal antibodies at a higher rate than did eitherof the Delaware E and GLS strain variants, resulting in more bursalatrophy and fewer protected birds. This study demonstrated that only thethreat posed by wild virulent Group 6 viruses, but also furtherreinforced the need for an effective vaccine against it.

One Group 6 IBDV isolate that is especially preferred for use in thepresent invention has now been identified as isolate 28-1. Isolate 28-1has substitution 318-D noted in FIG. 1, and in addition, has one or moreof the following additional substitutions in VP-2 of IBDV: 222-T, 249-K,254-S or N, 279-N and 286-I. Preferably, the 28-1 isolate will have atleast two of the additional substitutions noted, in particular 222-T and254-S or N, and more preferably at least three, four, or all five of theadditional substitutions noted. A suitable example has been depositedwith the ATCC under Accession Number PTA-5848 on Mar. 4, 2004. The virusisolate 28-1 differs from previous isolates by a combination of itsmolecular group classification (as set forth above), and also by thefact that it retains substantial reactivity with the basic panel ofmonoclonal antibodies that characterize the Delaware type variantviruses.

The Group 6 IBDV antigenic isolates, and in particular the 28-1 isolate,may be isolated using techniques available in the art. For example,bursae from infected chickens may be obtained from a commercial broilerflock. The virus may then be passaged in bursal tissue or other suitablemedia to establish a master seed virus. Further characterization by theskilled artisan may also be undertaken using available methods.

In a further aspect herein, the invention also comprises a vaccinecomposition containing one or more of the Group 6 IBDV antigenicisolates described. It is especially preferred that the IBDV antigenicisolate demonstrate significant immunogenic and safety characteristicswhen formulated into a vaccine composition. It is highly preferred thatthe qualities of the vaccine composition be sufficient to obtainregulatory approval and licensure thereof from the USDA. The 28-1isolate, and other isolates having substantially the same substantiveidentifying characteristics, are highly preferred for the vaccinecomposition. The Group 6 antigenic isolates herein described shouldoffer significant cross-protection against a wide range of IBDVchallenge variants, including without limitation, the Standard (STC), aswell as the Delaware and AL-2 variants, and variants from the Group 6molecular family, including 28-1. Protection against the Shelton 21, orS-21 variant strain of the Group 6 family is also contemplatedherein—the S-21 variant has the following substitutions in the VP-2region: 318-N and 321-E.

The vaccine composition of the invention may be formulated usingavailable techniques, preferably with a pharmacologically acceptablecarrier. For example, in one embodiment an aqueous formulation iscontemplated. Such formulations utilize water, saline, or phosphate orother suitable buffers. In still another embodiment, the vaccinecomposition is preferably a water-in-oil or oil-in-water emulsion. Alsocontemplated are double emulsions, often characterized aswater-in-oil-in-water emulsions. The oil may help to stabilize theformulation and further function as a vaccine adjuvant. Suitable oilsmay be selected by the skilled artisan, and can include, withoutlimitation, white oil, Drakeoil, squalane or squalene, as well as otheranimal, vegetable or mineral oils, whether naturally-derived orsynthetic in origin. In addition, the vaccine composition may containother suitable adjuvants available in the art. These can includealuminum hydroxide and aluminum phosphate, for example, as well as othermetal salts.

Additional excipients may also be included in the vaccine composition,such as surfactants or other wetting agents. Surfactants can include thesorbitan mono-oleate esters (TWEEN® series), as well as the ethyleneoxide/propylene oxide block copolymers (PLURONIC® series), as well asothers available in the art. Other compounds recognized as stabilizersor preservatives may also be included in the vaccine. These compoundsinclude, without limitation, carbohydrates such as sorbitol, mannitol,starch, sucrose, dextrin or glucose and the like, as well thepreservative formalin, for example.

The vaccine composition may also be formulated as a dry powder,substantially free of exogenous water, which may then be reconstitutedby an end user.

The vaccine composition may contain live virus, or live, but attenuatedvirus. Also contemplated is killed or inactivated virus for use in theinvention. In one embodiment, the virus utilized in the vaccinecomposition has been attenuated using serial passaging techniques. Wholeor partial antigenic isolate may be utilized. It is highly preferredthat whatever form of antigenic isolate is selected, the identifyingcharacteristics of the Group 6 family, in particular the antigenicfeatures hereinabove described, be retained in the featured vaccinevirus. Also contemplated for use herein is a recombinant vaccine, whichexpresses the nucleic acids, amino acid sequence, and/or proteins havingthe identifying structural characteristics of the Group 6, e.g. 28-1,antigenic isolates herein described.

The vaccine composition of the invention will contain an effectiveamount of IBD virus isolate for preventing or ameliorating InfectiousBursal Disease. In one embodiment of the invention, the vaccine willcontain from about 10^(2.0) to about 10^(6.0) EID₅₀ (“Egg InfectiousDose”) of isolate per dose. A dose is typically within the range ofabout 0.25 mL to about 2.0 mL per poultry animal, more preferably about0.5 mL to about 1.0 mL per animal.

The vaccine composition herein described may be administered with thenovel antigenic isolate set forth above, and in addition, may alsocontain one or more other IBDV variants as vaccine antigens, inparticular, those that are recognized as being efficacious against thedisease. For example, the Lukert strain, first isolated by Dr. PhilLukert of the University of Georgia at Athens and developed by SalsburyLaboratories, Inc., may be co-administered in an IBDV vaccinecomposition.

Other poultry antigens against other diseases may also be included andadministered with the vaccine composition of the invention. For example,vaccine antigens against chicken herpes virus, chicken anemia virus(CAV), Newcastle Disease virus and Infectious Bronchitis (IB) virus, aswell as reovirus antigens may be included as part of the vaccinecomposition of the invention. One or more reovirus antigens may beparticularly preferred as part of the vaccine composition of theinvention.

The invention is also directed to a method for inducing protectionagainst infection from Infectious Bursal Disease Virus. The methodinvolves administering to a poultry animal a vaccine compositioncontaining an antigen having the substantive identifying amino acidcharacteristics of an IBDV antigenic isolate as set forth in FIG. 1,i.e. an isolate from the now-identified family of Group 6 variants. Inparticular, the isolate 28-1 is preferred, and the isolate as set forthabove which is deposited under ATCC Accession # PTA-5848 on Mar. 4, 2004is particularly desirable.

The method of administration may be selected by the skilled artisan. Inovo administration is contemplated herein. For example, embryos may beinoculated, usually at about day 18-19. In addition, the vaccinecomposition may be administered to post-hatch, young (few days toseveral weeks old) chicks via drinking water, spraying or eye drops.Other methods wherein the vaccine composition of the invention isadministered parenterally, subcutaneously, peritoneally, orally,intranasally, or by other available means, preferably parenterally, morepreferably intramuscularly, in effective amounts according to a schedulewhich may be determined according to the time of anticipated potentialexposure to a carrier of the disease-causing Infectious Bursal DiseaseVirus, are also within the scope of the invention.

As set forth above, the invention is directed to novel IBDV antigenicisolates, vaccine compositions and methods for poultry. The term“poultry” is intended to encompass, without limitation, allcommercially-bred poultry animals, including chickens, ducks, geese,peafowl, bantam fowl, and the like.

In a further embodiment of the invention, there is also provided amarker vaccine for IBDV which utilizes the antigenic isolate as setforth in FIG. 1 (28-1) to distinguish between vaccinated andunvaccinated members of a flock, as well to distinguish vaccinatedanimals from those that may have been infected with one or more othervariants of the virus. The marker vaccine may also include a reagent kitfor testing animals. The reagent kit could include more or one antigenicisolates, such as 28-1, as well as antibodies which recognize and reacttherewith.

EXAMPLES

The following examples illustrate various preferred aspects of theinvention, but should not be construed as in any way limiting the fullscope thereof.

Example 1 Study Summary

Specific Pathogen Free (SPF) White Leghorn chickens were vaccinated atfive weeks of age with 1/50^(th) of a dose of prototype, inactivated,Infectious Bursal Disease Virus (IBDV)/Avian Reovirus combinationvaccines. The vaccines contained Avian Reovirus antigens and InfectiousBursal Disease virus, Lukert strain, with or without the addition of thevariant IBDV strains of bursal tissue origin (BTO). Test vaccines wereformulated into a 55% oil:45% aqueous ratio, 0.5 mL format. A competitorIBDV/Reo vaccine product was also administered at 1/50^(th) of a dose toa group of chickens.

The groups were bled for serum at 24 days post-vaccination and werechallenged with variant S-21, Delaware variant E or variant AL2 IBDVisolates at 28 days post-vaccination. Bursa to body weight ratios wererecorded at 10 days post-challenge.

Serologic and protection data from vaccinated chickens indicated thatthe antigen prepared from a variant isolate designated 28-1, and inparticular when added to IBDV Lukert ACL in an inactivated vaccine,significantly increased immunogenicity.

It was concluded that further vaccine development efforts should includethe 28-1 variant strain IBDV antigen.

Evaluation of Additional Variant IBDV Isolates as Inactivated VaccinesFractional Dose Study Background

A number of field isolates of Infectious Bursal Disease Virus (IBDV)were accessioned from cases in which a molecular typing system revealednovel or unique patterns to indicate viruses of variant antigenicity.This example was undertaken to examine and characterize a number ofthese isolates further for possible future development as either live,live (attenuated), or inactivated vaccines. The first step in thecharacterization was to evaluate the pathogenicity of the viruses. Basedon the results of the initial trial, selected isolates were used aschallenge viruses to determine which, if any, had the capability tobreak through immunity induced by current inactivated vaccines. Thethird study attempted to evaluate the use of two identified variantisolates in combination with IBDV Lukert ACL in inactivated vaccines,but it was invalidated by inadvertent pre-challenge virus exposure.

The currently described study was designed to evaluate the use of BTOantigens from alternate variant IBDV isolates in combination with IBDVLukert ACL in inactivated vaccines. Serologic response, as measured byELISA and serum neutralization (SN) assays, and protection against threevariant challenge strains were assessed.

Objective(s)

Compare the ability of prototype vaccines, prepared to include BTOantigen from IBDV variant isolates S-21, 15-4 (a Delaware strainvariant), 28-1 and ArkProvent, to immunize chickens. (S-21 andArkProvent are other molecular Group 6 virus isolates, but S-21 isconsidered a novel type as defined by a panel of monoclonal antibodies,and ArkProvent has not been so defined).

Evaluate the performance of the prototype vaccines in comparison to acompetitor product.

Materials and Methods Event Log

Test Day Activity 0 Vaccinated appropriate groups at 4 weeks of age 24Bled 15 birds per vaccine group and 20 birds from the nonvaccinatedgroups. 28 Challenged appropriate groups with IBDV challenge isolates 38Determined bursa to body weight ratios.

Animal Selection

Test Animals

Type: Gallus domesticus

Number: 400 (375 used in testing and 25 extras)

Serological Status: SPF

Age/Weight range: 3 weeks of age

Sex: Mixed

Breed: White Leghorn

Identification: Isolator cage cards

Source: HyVac

Preadmission/exclusion criteria: Only chickens in an apparently healthycondition at the time of inoculation were used. Any chicks which bytheir behavior or appearance were deemed to be in an ill-thriven statewere excluded from the study.

Housing and Care of Animals

The chickens were housed in negative pressure, modified Horsfal typePlexiglas isolators in the appropriate building of the test facility.They were provided feed and water ad libitum for the duration of thestudy. Standard poultry care procedures for the animal testing facilitywere used.

Test Vaccines Composition of Vaccine

A total of seven vaccines were tested in the study. One vaccine was anactual competitor's product (which contains at least one BTO antigencomponent) and the remaining six vaccines were prepared by Research &Development at Fort Dodge Animal Health, Fort Dodge, Iowa.

Test vaccines were comprised of inactivated IBDV and avian reovirusviral fluids formulated into a water-in-oil emulsion. IBDV Lukert ACLlot antigen was sourced from the Bioproduction facility, and was addedto five of the vaccines in an amount to yield a relative potency (RP) of1.0 as compared to the stock antigen. The Shelton 21, 15-4, 28-1 andArkProvent variant fluids were sourced from bursal tissue prepared inResearch & Development, and were added in an amount to yield a dose of10^(4.0) EID₅₀ per 0.5 ml. The fifth vaccine contained only the baseIBDV Lukert ACL antigen with no variant.

IBDV Lukert of chicklet origin, lot #XXXX-XX-XX was added to the sixthvaccine to yield 10^(6.5) TCID₅₀ per 0.5 mL dose. Additionally, theShelton 21 antigen was added at 10^(4.0) EID₅₀ per 0.5 mL.

Avian Reovirus antigens were sourced from TTPI-produced, ACL propagatedstocks and were added to all vaccines to yield 10^(6.7) TCID₅₀ per 0.5mL dose. The formulations were 55% oil:45% aqueous ratio with Arlacel 83and Tween 80 as emulsifiers, formulated for the 0.5 mL final volume perdose.

Source of Vaccine

Vaccines were prepared in the Fort Dodge R&D facility with the exceptionof the competitor's product which was obtained from a commercialdistributor.

Shipment of Vaccine

Vaccines were shipped prior to the initiation of the study on theregular intra-facility shuttle, and were transported and stored tomaintain a temperature of 2° C. to 7° C.

Serial Number

Prototype Fort Dodge vaccines were not identified by serial number. Theindividual bottles were labeled with the name indicated in the table ofTreatments below (TABLE 3).

Competitor's product—serial #1159011 (also contained reovirus antigen)

Quality Control Testing or EU Batch Certificate (if Available)

No testing was performed on the vaccines by the Quality ControlDepartment. Fort Dodge R&D performed pre-inactivation titration andcompleteness of inactivation testing on the variant BTO antigens as abasis for developing vaccine formulations.

Storage

Vaccines were stored at 2° C. to 7° C.

TABLE 3 EXPERIMENTAL DESIGN Route/ # Group Treatment Volume Challengebirds A Chicklet Lukert + S21 IM/0.01 mL See Challenge 45 Procedurebelow B Lukert ACL only IM/0.01 mL See below 45 C Lukert ACL + S21IM/0.01 mL See below 45 D Lukert ACL + 28-1 IM/0.01 mL See below 45 ELukert ACL + 15-4 IM/0.01 mL See below 45 F Lukert ACL + IM/0.01 mL Seebelow 45 ArkProvent H Competitor's product IM/0.01 mL See below 45 IChallenge control N/a See below 45 J Normal control N/a n/a 15

Vaccination

Chickens were vaccinated once, intramuscularly, in the breast. Thevaccine was delivered using a microsyringe with mechanical repeatingapparatus fitted to a 20 gauge needle, and 0.010 mL volume wasadministered to each chicken at 5 weeks of age.

Challenge and Observation Procedure

Appropriate dilutions of the Shelton 21, Delaware variant E and AL2challenge isolates were made in sterile tryptose phosphate broth toachieve a dose of 10^(3.0) to 10^(3.5) EID₅₀ per chicken. The challengewas administered by eye drop, using a pipettor and delivering 30 μl toone eye of each of 15 birds per group for each isolate. Challenge tookplace at four weeks post-vaccination.

At ten days post challenge, the groups were euthanized and bursa to bodyweight ratios were determined.

Challenge Table:

Challenge isolate Dose/Administration Challenge scheme Shelton 2110^(3.5) EID₅₀ per 0.03 mL eyedrop Once at 28 days to 15 birds/grouppost-vaccination Variant E 10^(3.5) EID₅₀ per 0.03 mL eyedrop Once at 28days to 15 birds/group post-vaccination AL2 10^(3.5) EID₅₀ per 0.03 mLeyedrop Once at 28 days to 15 birds/group post-vaccination

Sample Collection and Testing

Blood samples were collected at 24 days post-vaccination. The blood wasallowed to coagulate and the serum was decanted into separate tubes. Thesera was frozen and sent to Fort Dodge R&D for serologic analysis byIdexx ELISA (extended range) and in-house virus neutralization assays.

To determine bursa to body weight (B/BW) ratios, the chickens wereeuthanized and the body weight of each bird was recorded. For each bird,the bursa was excised in its entirety and was similarly weighed.

Samples of the bursae were also collected in neutral buffered formalinand archived.

Data Analysis

Geometric mean IBDV ELISA titers for the groups were calculated andcompared by one way ANOVA and Fisher's PLSD (α=0.05) after importationof the data into the Statview software package. ELISA data was alsoevaluated in terms of percent positive, wherein any sample with a signalto positive control (S/P) ratio greater than 0.20 was consideredantibody positive. (as specified by the ELISA assay kit manufacturer,Idexx). For serum neutralization data, geometric mean titers were alsocalculated, and for defining percent positive, any sample with areciprocal dilution of 20 or greater was considered antibody positive.

The B/BW ratio was calculated by dividing a recorded bursa weight by itsassociated body weight and multiplying by 1000. The normal controls wereused to establish a mean normal B/BW ratio, and a cutoff value wascalculated to be two standard deviations less than this mean. Any birdwith a B/BW ratio less than the cutoff value was considered affected bychallenge, thus not protected by vaccination. This analysis yielded dataof percent protected.

Group mean B/BW ratios were also compared by one way ANOVA (α=0.05) andFisher's PLSD after importation of the data into the software package. Agroup mean B/BW ratio that was significantly higher than the challengecontrols indicated protection was achieved in a group. A statisticallyhigher group mean B/BW ratio in one group compared to another group,indicated a significantly higher level of protection in that group.

Results and Discussion Serology

ELISA and serum neutralization assay data is reported in Table 4.

The challenge control and normal control groups remained free of IBDVantibody prior to challenge, validating the biosecurity of the study.

The vaccines containing Lukert ACL with the addition of BTO-sourcedvariant antigen elicited significantly higher ELISA GMT against IBDVthan did the Lukert ACL only group, but those titers were not alwayssignificantly higher than the nonvaccinated control groups. The LukertACL+15-4 vaccine elicited a significantly higher ELISA GMT than both thecontrol and Lukert ACL only groups, but only the 28-1 antigen increasedthe ELISA response at or above the level of the “benchmark” competitorproduct.

Serum neutralization (SN) assay GMT's were low in general and thus werenot evaluated statistically. In terms of percent SN positive, resultssimilar to those obtained by ELISA were observed. The control groupswere free of detectable antibody. The Lukert ACL only, ChickletLukert+S21, Lukert ACL+ArkProvent and Lukert ACL+S21 groups had only 1or 2 positives, and the Lukert ACL+15-4 and Lukert ACL+28-1 groups had 5positives.

Challenge Protection

Challenge protection was evaluated in two ways. First, as reported inTable 5, was by comparing group mean B/BW ratios. Table 5 includescomparisons split out by challenge isolate and for all challengescombined.

It is the “all challenges combined” data which may be most pertinent inevaluating the immunogenicity of the vaccines. This would be expected toprovide an overall view of protection against variants in general,including more recent isolates, which is the targeted objective of anyvaccine under development.

When the combined challenges data is considered, the mean B/BW ratios ofLukert ACL only, Chicklet Lukert+S21, Lukert ACL+S21 and LukertACL+ArkProvent groups were statistically equivalent to the challengecontrol group. This indicated that there was essentially no protectionafforded by vaccination with 1/50^(th) of a dose of these vaccines. TheLukert ACL+15-4 group had a mean B/BW ratio that was significantlyhigher than the challenge control group, indicating some level ofprotection was induced by this vaccine. The Lukert ACL+28-1 group had amean B/BW ratio that was significantly higher than the Lukert ACL+15-4group, indicating a still higher level of protection. The Competitor'sproduct vaccine fell between the Lukert ACL+15-4 and Lukert ACL+28-1groups, and was not significantly different than either of those groups.

The data was also evaluated in terms of percent protection as derivedfrom a cutoff value calculated from the nonchallenged, normal controlgroup of chickens, which is reported in Table 6. Again, the data derivedby combining all of the challenges is probably most pertinent. Thisevaluation essentially gave the same results as the group mean B/BW data(Table 5) in that the Lukert ACL+28-1 and the competitor's productvaccine elicited higher levels of protection than the others, with theLukert ACL+28-1 group giving the numerically highest level ofprotection.

Discussion

One of the primary objectives of the study was to determine if othervariant isolates combined with IBDV Lukert ACL would yield moreantigenic vaccines as determined by serologic and protective responses.That objective was essentially achieved in the study as it wasdetermined that supplementation of vaccine with antigen derived from the28-1 isolate resulted in more immunogenic vaccines. When formulated intoprototype vaccines at equivalent dose and administered in equivalentfractional dose volumes by equivalent routes, the 28-1 supplementedvaccines elicited significantly greater ELISA and protective responsesthan other vaccines. The second objective of the study was to evaluatethe various prototype vaccines in comparison to a competitor productwhich performed well in previous studies. By all means of evaluationexcept percent SN response, the vaccine supplemented with 28-1 BTOantigen met or exceeded the performance level of the competitor'sproduct vaccine. Hence the 28-1 antigen is highly suitable for furthervaccine development.

The 1/50^(th) of a normal dose given in this study was designed to givelowered protection levels such that differences between the vaccinescould be elucidated.

CONCLUSIONS

The inclusion of 28-1 isolate BTO antigens significantly increasedserologic and protective responses elicited by prototype vaccines.

The inclusion of 28-1 isolate BTO antigen yielded a prototype vaccinewhich performed at, or surpassed the level of the competitor's productwhen serologic response and protective response against variant IBDV wasconsidered.

TABLE 4 SEROLOGIC RESPONSE INDUCED BY PROTOTYPE AND CURRENTLY-LICENSEDINACTIVATED VACCINES WITH AN IBDV COMPONENT. ELISA response SN responseGeometric Geometric Treatment mean titer* % positive mean titer %positive Chicklet  15^(a,b)  0% (0/15) 1  7% (1/15) Lukert + S21 LukertACL only  5^(a)  7% (1/15) 1 13% (2/15) Lukert ACL +  32^(b)  0% (0/15)2 13% (2/15) S21 Lukert ACL + 589^(d) 60% (9/15) 4 33% (5/15) 28-1Lukert ACL +  66^(c) 20% (3/15) 4 33% (5/15) 15-4 Lukert ACL +  32^(b)20% (3/15) 1  7% (1/15) ArkProvent Competitor 491^(d) 73% (11/15) 23 73%(11/15) Challenge control  27^(b)  0% (0/15) 1  0% (0/15) Normal control 10^(a,b,c)  0% (0/5) 1  0% (0/5) *Means with the same superscripts arestatistically equivalent by one-way ANOVA and Fisher's PLSD (α = 0.05)

Protection Against Variant IBDV Challenge Induced by Prototype andCurrently-Licensed Inactivated Vaccines with an IBDV Component asMeasured by Group Mean Bursa to Body Weight Ratios

TABLE 5 Mean group B/BW ratio* All Challenge strain challenges TreatmentS-21 Variant E AL-2 combined Chicklet Lukert + S21 0.92^(a) 1.11^(a)0.97^(a,b) 1.00^(a) Lukert ACL only 0.96^(a) 1.16^(a,b,c) 0.96^(a)1.03^(a) Lukert ACL + S21 1.06^(a,b) 0.98^(a) 1.02^(a,b) 1.02^(a) LukertACL + 28-1 1.17^(a,b,c) 2.24^(d) 1.77^(c) 1.73^(c) Lukert ACL + 15-41.40^(b,c) 1.52^(b,c) 1.04^(a,b) 1.32^(b) Lukert ACL + ArkProvent1.01^(a,b) 1.03^(a) 0.88^(a) 0.97^(a) Competitor 1.49^(c) 1.62^(b,c)1.44^(b,c) 1.52^(b,c) Challenge control 0.90^(a) 0.99^(a) 1.01^(a,b)0.97^(a) Normal control 4.17^(d) 4.17^(e) 4.17^(d) 4.17^(d) *Meanswithin columns with the same superscripts are statistically equivalentby one-way ANOVA and Fisher's PLSD (α = 0.05)

Against Variant IBDV Challenge Induced by Prototype andCurrently-Licensed Inactivated Vaccines with an IBDV Component asMeasured by Percent Protection in Reference to a Calculated Cutoff Value

TABLE 6 Percent Protected* All Challenge strain challenges TreatmentS-21 Variant E AL-2 combined Chicklet Lukert + S21 0 0 0 0 Lukert ACLonly 0 7 0 2 Lukert ACL + S21 7 7 0 4 Lukert ACL + 28-1 7 60 27 31Lukert ACL + 15-4 13 13 0 9 Lukert ACL + 7 7 0 4 ArkProvent Competitor20 13 13 16 Challenge control 0 0 0 0 Normal control n/a n/a n/a 100*Percent protected was based on a cutoff value using the normal controlgroup. Normal control group mean bursa to body weight ratio (4.17) minustwo standard deviations (2.23) = calculated cutoff (1.94). A bird whosebursa to body weight ratio exceeded 1.94, when necropsied 10 dayspost-challenge, was classified as protected.

Examples 2 and 3

As depicted in FIGS. 3 and 4, the vaccine composition of the inventionwas further compared in broiler progeny challenge studies. In each fieldtrial, the standard program consisting of two shots of commercialvaccines was compared to a program whereby the vaccine composition ofthe invention replaced one of the commercial vaccines. The broilerbreeder sources were 38-44 weeks of age when fertile eggs were collectedfor the study. The results are graphed in terms of percent protection.In FIGS. 3 and 4, the results on the right represent the vaccine programcontaining the composition of the invention.

While the invention has been described in each of its variousembodiments, it is expected that certain modifications thereto may beundertaken and effected by the person skilled in the art withoutdeparting from the true spirit and scope of the invention, as set forthin the previous description and as further embodied in the followingclaims.

1-18. (canceled)
 19. An Infectious Bursal Disease Virus (IBDV) ViralProtein-2 (VP-2) antigen comprising the amino acid sequence of SEQ IDNO:1 and further comprising two or more amino acid substitutions,wherein the first amino acid substitution is G to D at amino acidposition 318 and wherein said first amino acid substitution is the onlyamino acid substitution within the major hydrophilic peak B of the VP-2amino acid sequence, and wherein the second amino acid substitution is254-N.
 20. A vaccine composition comprising the IBDV VP-2 antigen ofclaim
 19. 21. The vaccine composition of claim 20, further comprising apharmacologically acceptable carrier.
 22. The vaccine composition ofclaim 21, wherein said carrier is an aqueous carrier.
 23. The vaccine ofclaim 21, wherein said carrier is an emulsion.
 24. The vaccine of claim22, wherein said emulsion is a water-in-oil emulsion.
 25. The vaccinecomposition of claim 20, further comprising at least one additionalvaccine antigen.
 26. The vaccine composition of claim 25, wherein saidat least one additional vaccine antigen is selected from the groupconsisting of chicken herpes virus antigens, chicken anemia virus (CAV)antigens, Newcastle Disease virus antigens, Infectious Bronchitis (IB)virus antigens, reovirus antigens, and Infectious Bursal Disease Virus(IBDV) variant antigens.
 27. A method for inducing protection againstinfection from Infectious Bursal Disease Virus, said method comprisingadministering to a poultry animal the vaccine composition of claim 20.28. The method of claim 27, wherein said vaccine is administered to saidpoultry animal in ovo.
 29. The method of claim 27, wherein said poultryanimal is a young post-hatch chick.
 30. An Infectious Bursal Diseasevirus comprising a Viral Protein-2 (VP-2), wherein said VP-2 comprisesthe amino acid sequence of SEQ ID NO:1 and further comprising two ormore amino acid substitutions, wherein the first amino acid substitutionis G to D at amino acid position 318 and wherein said first amino acidsubstitution is the only amino acid substitution within the majorhydrophilic peak B of the VP-2 amino acid sequence, and wherein thesecond amino acid substitution is 254-N.
 31. A vaccine compositioncomprising the Infectious Bursal Disease virus of claim 30 and apharmacologically acceptable carrier.
 32. The vaccine composition ofclaim 31, wherein said Infectious Bursal Disease virus is a live virus.33. The vaccine composition of claim 32, wherein said Infectious BursalDisease virus is a live-attenuated virus.
 34. The vaccine composition ofclaim 33, wherein said live-attenuated virus has been serially passaged.35. The vaccine composition of claim 31, wherein said Infectious BursalDisease virus is a killed-inactivated virus.
 36. The vaccine compositionof claim 31, wherein said carrier is an aqueous carrier.
 37. The vaccinecomposition of claim 31, wherein said carrier is an emulsion.
 38. Thevaccine composition of claim 37, wherein said emulsion is a water-in-oilemulsion.